Showing papers in "Biotechnic & Histochemistry in 1971"

Xylenol orange, a fluorochrome useful in polychrome sequential labeling of calcifying tissues.

Abstract: Xylenol orange is fixed in newly formed calcified tissues where it remains until removal of the bone mineral. It may be visualized in undecalcified histological sections in a similar way to the tetracyclines by means of its fluorescence. The fluorescence contrasts to that of known fluorescent dyes. Intravital bone labeling by Xylenol orange is achieved by parenteral administration of the dye. A dose of 90 mg/kg given as a 3% aqueous solution was found suitable. The general toxicity, local effect on calcification and resistance of the fluorescence to histological chemicals and long term excitation were assessed. It is noteworthy that the fluorescence of Xylenol orange is found exactly at the same site as that of tetracyclines, fluoresceins and Calcein blue. Xylenol orange is thus suitable for single or for polychrome sequential labeling.

The Use of a Chrome Alum-Gelatin (Subbing) Solution as a General Adhesive for Paraffin Sections

Abstract: The adhesion obtained from a chrome alum-gelatin solution has been found far superior to results given by widely used general adhesives (Haupt's gelatin and Mayer's egg albumen) for paraffin sections. The subbing solution, which consists of 5.0 gin gelatin and 0.5 gm chrome alum per liter of water, is easier to apply and gives more consistent results. Sections affixed to subbed slides are resistant to removal by acids and bases: 1.0 and 0.1 N HCl or H2SO4, 1 M H3PO4, 5% oxalic and trichloroacetic acids, 1% and 10% lactic acid, 1.0 and 0.1 N NaOH or NH4OH, and other fluids and solutions such as organic solvents, water, hypochlorite, KMnO4 and thiosulfate. The applied adhesive is virtually unstained by many stains, including hematoxylin, eosin, fast green, safranin, PAS, Sudan IV and Mallory's triple stain. The only treatment yet found to detach affixed section in less than 6 hr is immersion in 5% trichloroacetic acid for 15 min at 100 C. The concentration of gelatin and chrome alum in the solution recommen...

Grimelius' Silver Stain for Endocrine Cell Granules, as Shown by Electron Microscopy

Abstract: The silver impregnation method of Grimelius has been applied to 100-150 μ thick sections of tissues fixed 2 hr to 1 mo in mixtures containing formaldehyde, glutaraldehyde or picric acid. After silvering, the sections (partly postfixed in 1% OsO4, for 0.5 hr) were processed for electron microscopy. Endocrine granules of pancreatic A cells, enter-ochromaffin and some nonenterochromaffin cells of the gastrointestinal mucosa, thyroid C cells and adrenal medullary cells were found to be selectively stained by silver grains 10-30 nm in diameter, either as a peripheral “halo” or covering the entire granule. At least in some cells, the reactive material should not be identified with the hormonal products known to be stored in the granules.

Basic two-dye stains for epoxy-embedded 0.3-1 sections.

Abstract: Dyes used in the 3 methods recommended are: I, thionin and acridine orange (T-AO); II, Janus green and Darrow red (JG-DR); III, methyl green and methyl violet (MG-MV). The first 2 methods were two-solution stains, applied in sequence; the third, required only one solution since methyl violet is present in commercial methyl green. Staining solution and timing was as follows: Method I. 0.1% thionin in a 45% ethanolic solution of 0.01 N NaOH, 5 min at 70 C; rinsing in water and followed by 1 min in a 1% aqueous solution of acridine orange made up in 0.02 N NaOH, also at 70 C, then washed, and dried on slides. Method II. 0.5% Janus green in aqueous 0.05 N NaOH, 5 min at 70 C; rinsing in water then into 0.5% Darrow red in 0.05 N NaOH (aq.), 2 min at 70 C., washing, and drying on slides. Method III. 1% methyl green (commercial, unpurified) in 1% aqueous borax for 15-20 min at 20-25 C, washing and attaching to slides. All staining was performed by floating the sections on the staining solutions, all drying at 70...

Embedding Media for 1-2 Micron Sectioning. 3. Hydroxyethyl Methacrylate-Benzoyl Peroxide Activated with Pyridine

Abstract: A hydroxyethyl methacrylate (HEMA) monomer medium containing 2-butoxyethanol as the plasticizer requites only one stock solution consisting of: 50 ml of 94 or 96% HEMA containing 200 ppm inhibitor (Rohm and Haas, Philadelphia) is mixed with 12 ml 2-butoxyethanol; 025 gm benzoyl peroxide is added and permitted to dissolve at 20-25 C This mixture is activated by the addition of 08-10 ml of pyridine Polymerization of the activated mixture is initated in 15-20 hr at 25 C and in 2-4 hr at 50 C; polymerization of the mixture is complete in 2-3 days and 3-6 hr, respectively The activated monomer mixture is stable at temperatures below 18 C; hence infiltration of tissues may be extended to 7-10 days by keeping the mixture at 0 to -40 C

The Pearson silver-gelatin method for light microscopy of 0.5-2 plastic sections.

Abstract: Sections 0.5-2 μ thick of liver, kidney, lung, cartilage and brown fat embedded in Maraglas, Araldrite, Epon and Spurr's medium were deplasticized in alcoholic NaOH for 15 min. Following several alcohol rinses, the tissues were exposed to 2% AgNO3 at 50 C for 1-2 hr, and then developed in a solution containing 3% gelatin, 40 ml; 2% AgNO3, 10 ml; and 1% hydroquinone, 4 ml. Sections were then toned in 1% gold chloride (several dips), washed in water for 5 min and dipped in 2% oxalic acid. After a brief rinse in water the sections were placed in 5% Na2S2O3 for 5 min, washed in water for 5 min, dehydrated in alcohol, cleared and covered. Compared to similar sections retained in plastic and deplasticized sections stained in the routine manner with 1% toluidine blue, silver impregnated sections of all tissue free of plastic, displayed greater clarity of cellular detail. Especially clear were mitochondria of liver and brown fat cells. Type II cells of the lungs were exceptionally prominent as were podocyte foot ...

Basic fuchsin in acid alcohol: a simplified alternative to Schiff reagent.

Abstract: The use of Schiff reagent to demonstrate polysaccharides (after prior periodic oxidation) and nucleic acids (after prior acid hydrolysis) is unnecessary since the same results are obtained by substituting a 20 min staining in a 0.5% w/v solution of basic fuchsin in acid alcohol (ethanol-water-concentrated HC1, 80:20:1) followed by a rinse in alcohol. The shade of the basic fuchsin staining is a little yellower than that achieved with Schiff reagent but the selectivity, light fastness, response to different fixatives, and to prior histo-chemical blocking of the tissue section were much the same for the two methods. The need for prior oxidation or hydrolysis and the inhibitory effect of aldehyde blocking techniques indicate that basic fuchsin, like Schiff reagent, reacts with aldehyde groups. Infrared studies indicate that for cellulose the reaction product is an azomethine.

Use of gallocyanin as a myelin stain for brain and spinal cord.

Abstract: Tissue fixed in 10% formalin, formol saline, CaCO3 or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22–25 C. Mordanting 30–60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1–2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na2CO3, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5–10 mi...

Section thickness and grain count variation in tritium autoradiography.

Abstract: H2 styrene-glycolmethacrylate is a suitable standard source for autoradiographic model studies. When it is used for quantitative autoradiography, the coefficient of variation for grain counts is minimal at a section thickness of 2.5 μ and greater. Variation increases with progressively thinner sections. When sections of the same thickness are mounted on separate slides, there is no significant variation in grain counts at the 95% confidence level when the slides are processed simultaneously.

Analysis and purification of biological stains by gel filtration.

Abstract: Some sixty biological stains of widely varying type have been subjected to gel filtration chromatography in columns of Sephadex LH-20 (Pharmacia, Uppsala) resin swollen by dimethylformamide saturated with NaCl. Most dyes contained more than one coloured constituent. Measures of their molecular weights were obtained. The use of the method for analysis was as effective as other common chromatographic or electrophoretic procedures but was technically simpler. Preparative use of the method merely involved using a larger column of gel, though occasionally use of ethanol as solvent gave better resolutions and did provide easier recovery of separated dyes.

Methyl green-pyronin for staining autoradiographs of hydroxyethyl methacrylate-embedded lymphoid tissue.

Abstract: Cells in the spleen in DNA-synthesis were labelled with tritiated thymidine. Tissue was fixed for 12 hr in 10% neutral formalin, washed for 4 hr in tap water and dehydrated through 70% and absolute ethanol. The tissue blocks were infiltrated overnight with a mixture consisting of glycol methacrylate, 80 ml; polyethylene glycol 400, 12 ml; and benzoyl peroxide, 0.27 gm. Specimens were cast in BEEM capsules with the final embedding medium consisting of 42 parts of the infiltration medium and 1 part of an acceleration mixture. This mixture consisted of N,N-dimethylaniline, 1 part and polyethylene glycol 400, 15 parts. The blocks hardened in 30 min and were sectioned with an ultramicrotome fitted with glass knives. Sections were coated with Ilford K5 liquid emulsion and exposed for 2 wk. Methyl green-pyronin staining of autoradiographs was carried out at pH 4.1 in acetate buffer containing 0.5% methyl green (Allied Chemicals) and 0.2% pyronin GS (Chroma). Staining was for 30-60 min, after which sections were ...

Demonstration of the microvasculature of the spinal cord by intravenous injection of the fluorescent dye, thioflavine S.

Abstract: In the past, thioflavine S has been used for visualizing blood vessels and patterns of blood flow (Schlegel 1949; Schlegel and Moses 1950; Oliver et al. 1951). Methods employed have involved an intravenous injection of the dye, immersion of hand-cut sections in glycerol and examination of sections under incident Wood's light. With improved techniques it is possible to obtain microtome-cut sections and to use a more intense light source for enhancing fluorescence and resulting visualization of small vessels. Occlusion of arterioles by undissolved dye particles is prevented by ultracentrifugation of the solution to be injected.

Epidermal and Cuticular Mounts of Plant Material Obtained by Maceration

Abstract: Mounts of leaf and stem epidermises or bare cuticles, useful in both general anatomical and specialized phylogenetic studies, can be prepared by a maceration process using Jeffrey's solution (equal volumes of 10% aqueous CrO3 and 10% HNO3). Leaves, including those of conifers, and stems with cuticles thick enough to maintain integrity when isolated are amenable to this process. Dried specimens are hydrated by boiling in water; fluid-preserved specimens are washed thoroughly in water; fresh specimens need no pretreatment. Specimens are cut to a convenient mount size and trimmed so as to allow adequate and even penetration of the macerating fluid. Laminar leaf segments are left with one edge untrimmed so that upper and lower epidermises remain contiguous. Cylindrical leaf and stem segments are slit lengthwise through about half their thickness. Specimens are macerated in Jeffrey's solution for 1 to 4 days until unwanted tissues are loosened and easily freed from the epidermis (or bare cuticle, if that is de...

Rapid Chromosome and Sex-Chromatin Staining with Pinacyanol

Abstract: This simple and reliable 10-min procedure for producing uniformly and intensely stained, as well as fade-resistant, chromosome and sex-chromatin preparations uses pinacyanol chloride as the dye. Slides are extracted in 5 N HC1 at 20-23 C for 2 min, washed in running tap water for 2 min, stained in 0.25% pinacyanol chloride solution (made up in 70% methanol) for 45 sec, differentiated in Wright's buffer solution (pH 6.4-6.5) for 45 sec, washed in running tap water for 5 sec, dehydrated in 2 changes, 1 min each, of absolute tertiary butanol, cleared in 3 changes of xylene, a minimum of 30 sec each, and mounted in a neutral synthetic resin such as Permount.

Effects of Ethanol on the Metachromatic Reaction of Toluidine Blue O

Abstract: Ethanol abolishes the metachromatic reaction of toluidine blue O with un-combined chromotropes but not when they are in association with protein. The green colour obtained in metachromatic regions is established as not due to any green impurity of the dye by chromatographic analysis but due to the fluid dehydrants combining with the dye as dye-organic solvent mixture showed green. The loss of metachromasia is not due to a dehydration effect of ethanol alone for the following reasons: (i) Stained samples of chromotropes dried in vacuuo continued to retain the metachromatic colour, (ii) Although other dehydrating agents likewise abolished the metachromasia, alcohols which have very slight affinity to water also abolished it, (iii) Ethanol does not abolish metachromasia produced in an acid mucopolysaccharide-protein complex. This has been suggested as due to the inability of ethanol to separate the dye from such compounds and to bring about a shift to green.

Saponification of methylated mucosubstances at low temperatures.

Abstract: A fresh 1% solution of KOH in 70% ethanol in 2 hr at 2-4 C restores basophilia to methylated acid mucosubstances satisfactorily without detaching or damaging tissue sections. A 0.5% solution of Ba(OH)2 under the same conditions gives results nearly as good, but NaOH and KMnO4 are unsatisfactory.

Preparation of Tissue for Scanning Electron Microscopy: Freeze-Fracturing as a Technique for Enhancing Visibility of Structural Relationships

Abstract: Retinas from 3-wk-old mice were prepared for scanning electron microscopy by freezing in Freon 12 at —160 C, cleaving, and then freeze-drying. Preparations were shadowed, and produced clean surfaces which showed well-preserved detail in three dimensions.

Accurate placement of ultrathin sections on grids; control by sol-gel phases of a gelatin flotation fluid.

Abstract: A 3% solution of gelatin in a petri dish at 25-30 C. provides a liquid, viscous surface upon which ultrathin sections can be floated. On cooling, the gelled substratum immobilizes the sections, allowing grids to be placed on them with any desired grid-to-section orientation. When the gelatin is remelted, the sections remain attached to the grids. After draining, traces of gelatin adhering to the grids are removed by flotation (section side down) for 30 min on 2% acetic acid at 60 C. This is followed by flotation for 3-5 min on Tris buffer, pH 7.1, and then on distilled water for 30 min—both treatments at 60 C. The technique is particularly useful for mounting serial sections.

Photographic Mapping of a Tissue Surface to Locate Fields for Electron Microscopy; Mouse Lung

Abstract: Precise sampling from whole lobes of mouse lungs fixed in the inflated state and embedded in epoxy resin can be not only feasible but also efficient. A 1 μm section is cut from an embedded lobe with a rotary microtome and a steel knife. This section is stained and photographed, and from it a 35 × enlarged print is prepared. A grid of transparent plastic scored with 35 mm squares, lettered vertically and numbered horizontally, is superimposed over the photograph. The area chosen for electron microscopy thus becomes identifiable by a letter-number designation obtained from the grid. This area is then located by light microscopy on a 2 mm slice taken from the block from which the 1 μm section was cut, by use of oblique illumination and the calibrated mechanical stage of the light microscope. A block of 1.3 mm diameter is removed for electron microscopy from the tissue by a rotatable circular spring-loaded punch screwed into the objective turret of the microscope. The removed cylinder is mounted on a metal st...

Methyl green as a cartilage stain; human embryos.

Abstract: The development of this technique derived from a need to demonstrate the sites of developing cartilage in the human embryo. The basic procedure was that of Yntema (1970), which used methyl green on turtle embryos. However, the thickness of the soft tissues in human embryos of 90-125 mm crown-rump (CR) length and the opacity caused by pigments in this tissue when the specimen was cleared necessitated the modification of the original procedure; specifically, the introduction of bleaching. The altered procedure is given below.

Reducing Breakage of Metaphase Plates in Feulgen-Stained Root Tips of Wheat; Ice-Water Storage Before Squashing

Abstract: Cytogenetic studies with aneuploid series in wheat usually involve a large number of chromosome counts. The reliability of results and the speed of this work are limited by several factors: (1) method of pretreatment for contraction and spreading of chromosomes, (2) the staining procedure, (3) the method of maceration, and (4) the technique of squashing. These determine the final quality of squash preparations.

Albumen Embedding and Individual Mounting of One or Many Mammalian Ova on Slides for Fluid Processing

Abstract: For studies of ova with the light or electron microscope, as well as for autoradiographic and histochemical studies, these cells need to be sectioned. The handling of individual, often hard-to-obtain, cells through fluid processing by micropipettes is time-consuming and can easily cause damage or loss of valuable specimens. A number of interesting methods have been described for handling ova or free-floating cells. In these methods cells are commonly handled in containers with fine-mesh, wire cloth bottoms, when a number of cells are involved. Unfortunately they all require special equipment not readily or easily available (Buchanan 1965; Rinaldi et al. 1966; Izquierdo 1967; Shands 1968). In our method, egg white provides a supporting matrix for mouse ova and allows one or several specimens to be mounted on a slide.

Cold and chemical pretreatments to aid chromosome counts in a grass leaf squash technique incorporating hot pectinase maceration.

Abstract: Leaf buds, comprising the basal 3-5 mm of the youngest leaves attached to short stems, were dissected out of fast-growing young tillers of certain grasses, including Festuca, Lolium and Phalaris spp. and various hybrids. They were kept overnight in distilled water at 0-2 C, treated in a mixture of equal parts by volume of saturated aqueous solutions of 5,7-dibromo-8-hydroxyquinoline containing a surfactant (Tween 80), and 1-bromo-naphthalene for 3-4 hr at 0-2 C, and fixed in Newcomer's fluid. The rinsed samples were hydrolysed in 1 N HCl for 8 min at 60 C and Feulgen stained for 1 hr. After rinsing, the buds were macerated in a filtered 3% solution of Pectinol 100-D (Rohm and Haas) in 0.1 M acetate buffer at pH 4.5 for 10 min at 60 C. Squashes were made in 45% acetic acid. The combined cold and chemical pretreatments resulted in strongly contracted, easily counted metaphase chromosomes, while intact cells with full chromosome complements were more readily retained during squashing after enzyme maceration ...

Difficulties in Ferrous Iron Mordant Hematoxylin Staining and Some Hematoxylin Substitutes

Abstract: A gradual deterioration of intensity of sequence ferrous sulfate hematoxylin staining was traced, after elimination of hematoxylin quality as a cause, to a deterioration of the metal salt, associated with caking of the crystals. Fresh samples were also partly caked and ineffective. Ferrous ammonium sulfate was found also subject to the same deterioration. Ferrous chloride freshly prepared as a 1 M solution from iron wire under anaerobic conditions at biweekly intervals proved to be satisfactory as a mordant source. Of several other mordant dyes tested: gallein, brazilin and chromoxane pure blue B were the best, but none was equal to good hematoxylin.

Keratin in the EGG shells of amphistomes; histochemical differentiation from quinone tanned protein in other trematoda.

Abstract: Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H2SO4 for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H2SO4 and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.

Sequential use of Wright's and Ziehl-Neelsen's stains for demonstrating phagocytosis of bacterial spores.

Abstract: Nongerminating spores, germinating spores, and vegetative cells of Clostridium botulinum type A were observed during phagocytosis in the peritoneal fluid of white mice. Since phagocytes are easily ruptured by heat, the method described avoids heating, as this has been employed in conventional spore staining methods. A thin smear of the fluid is air dried on the slide for 2 hr, and stained by Wright's method: stain, 2 min; dilution water, 2 min; and rinsed; then in 0.005% methylene blue for 30 sec, and rinsed. This is followed by Ziehl-Neelsen's stain for 3-4 min and destained with 1: acetone-95% ethanol for 10 sec. The slide is rinsed, and Wright's staining repeated: stain 1 min, dilution 2-3 min; and thereafter washed about 5 ml of Wright's buffer. Blotting and air drying completes the staining. Non-germinating spores stain light red with a red spore wall, germinating spores are deep red throughout, vegetative cells are blue, and leucocytes show a dark purple nucleus and light blue cytoplasm.

Sandwich embedding of multiple layers of muscle tissue culture for serial frozen sectioning.

Abstract: Serial sections of fresh-frozen skeletal muscle fibers are necessary for demonstrating comparative histochemical relationships between oxidative enzymes and either myofibrillar adenosine triphosphatase or glycolytic enzymes in different fiber types (Dubowitz and Pearse 1960, Engel 1962).

Chemical and spectrometric evaluation of glycogen after routine histological fixatives.

Abstract: A detailed comparison of fixatives used for the demonstration of glycogen has been based on chemical assay and microspectrophotometry. Rat liver containing known amounts of glycogen was fixed in formol alcohol, Rossman's fluid, 10% neutral formalin, Bouin, Helly, SUSA, and Zenker's fluid at 4 C and 18 C. Chemical assay was carried out before and after fixation and paraffin sections were prepared from the fixed material. Sections were stained with PAS and the silver methenamine method. Visual examination was carried out with a comparison microscope and quantitative estimations on PAS-stained sections were performed by scanning microspectrophotometry. The histochemical methods were compared with the chemical results obtained from the same tissue and a reasonable degree of correlation between the sets of results was observed. Cold formol alcohol and cold Rossman's fluid preserved the most glycogen and Zenker and SUSA fixation preserved the least. Cold formol alcohol was the only fixative that preserved thres...